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Indian Journal of Plant Sciences ISSN: 2319–3824(Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm
2015 Vol.4 (1) January-March, pp.5-10/Chaturvedi et al.
Research Article
EVALUATION OF FREE RADICAL SCAVENGING ACTIVITIES OF
SIDA RHOMBIFOLIA EXTRACTS
*
Chaturvedi P., Kwape T.E. and Fulukani I.
Department of Biological Sciences, University of Botswana, Gaborone, Botswana
*
Author for Correspondence
ABSTRACT
In the present study, different extracts of aerial part of the plant using solvents like 70% methanol, 100%
methanol, 100% ethanol, 100% chloroform and 100% hexane were tested for DPPH and ABTS free
radical scavenging activity along with the measurement of their total phenol content. Results showed that
the inhibition DPPH radical was dependent on the concentration of the extract. The extent of percentage
inhibition also increased from a low polarity solvent extract to a high polarity solvent extract. Thus the
inhibition showed the pattern of 70% methanol > 1000% methanol> 100% >100% hexane respectively.
The standards gallic acid displayed 100 % DPPH inhibition at the dose 5000ug/ml. Highest ABTS
scavenging activity was seen in methanol extract having IC50 (µg/ml) of 50. Gallic acid showed IC50
(µg/ml) value as 25. Total phenol content showed the same pattern being highest in 70% methanol extract
(560g GAE/g)) and lowest in 100% hexane extract (260mg GAE/g). Thus all SR extracts from solvent
extraction of aerial parts showed free radical scavenging activity. Activity decreases as the polarity of
solvent decreases. Hence extract from most polar solvent had greater activity than extract from less polar
solvent. Free radical activity could be due to the phenol content.
Keywords: Free Radicals, Anti-oxidants, Phenol Content, DPPH, ABTS, Oxidative Stress
INTRODUCTION
Modern life style imposes various detrimental impacts on the human population. Present day population
relies on taking fast food because of their busy schedule. They are used to take pain killers for relief of
pain, sedatives and alcohols to overcome the mental stress. All these result into oxidative stress
According to Karbownik and Lewinski (2003), oxidative stress is involved in many diseases, such as
atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, and chronic
fatigue syndrome. Oxidative stress depletes the anti-oxidants present in the body and become the
causative factors for above mentioned physiological disorders (Paul et al., 2011). Therefore, there is a
need to supplement the anti-oxidant system of our body. Best way to get rid of the imbalance is the
supplementation of food with the diet rich in anti-oxidants. Green vegetables, leaves and fruits are the
natural source of anti-oxidants. Apart from them, traditional medicines also play a significant role in
alleviation of the oxidative stress conditions. Sida rhombifolia (SR) is one of those traditional medicines.
In traditional African medicine decoctions of the roots and leaves are widely used as emollient (Burkill
1997). The leaves or the leaf sap are applied to the skin as an antiseptic and to treat abscesses, ulcers and
wounds (Balkrishanan et al., 2008). In Cameroon, macerated extract of leaves of the plant is used as anti
hypertensive and to cure diarrhoea (Naumi et al., 1999). Our previous study reported the hypoglycemic
and hypolipedemic effects of extracts of aerial parts of SR (Chaturvedi et al., 2009).
SR is a short-lived perennial subshrub (woody stem and herbaceous branches) commonly growing to 60
cm, but sometimes reaching 1.5 m in height. It is found throughout Botswana. The plant is predominantly
found in eastern countries such as India where it is used in Ayurvedic system of medicine. Dhalwal et al.,
(2007) has reported the anti-oxidant properties of leaf, root and whole aerial parts of SR. As per his
report, whole aerial part could not prevent lipid peroxidation in liver and brain homogenate because of its
little antioxidant properties. Plants of the same species growing in different environment may not have
same potency with reference to their effect on particular biological system. Hepatoprotective effects of
water extract of aerial part have already been reported by Rao et al., (1997) and the protection is rendered
only by the anti-oxidant effect. Even in the Ayurvedic system of medicine, whole aerial part is being used
© Copyright 2014 | Centre for Info Bio Technology (CIBTech)
5
Indian Journal of Plant Sciences ISSN: 2319–3824(Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm
2015 Vol.4 (1) January-March, pp.5-10/Chaturvedi et al.
Research Article
for different formulation for different ailments. Because of these reasons, it was decided that the different
extracts of the whole aerial part of SR should be studied for their anti-oxidant properties. Therefore the
present study has been aimed to carry outthe free radical scavenging potency as well as the total phenol
content of different extracts obtained after extraction with solvents of different polarities.
MATERIALS AND METHODS
Collection of Plant Materials
The aerial parts of SR were collected along the roadside and by the construction sites in the University of
Botswana main campus. The whole plant was first washed with tap water and rinsed with distilled water
and allowed to dry. The dried plant material was then ground to powder using a blender. Different types
of extracts were then prepared from this powder SR material.
Preparation of 70% Methanol, 100% Methanol, 100% Chloroform and 100% Hexane Extracts
Soaking the plant in a solvent, such as water, methanol and chloroform, dissolves the active ingredients
and separates them from the plant material. The powered plant (375grams) was first soaked in 1000ml of
hexane. The plant material was allowed to soak in hexane for 24 hours, after 24 hours, Hexane soaked
with the plant extract was then filtered in an empty bottle. Chloroform was added to the same bottle
containing the powdered plant material, it was allowed to soak for the minimum of 24 hours before
filtering into another empty bottle. The same procedure was carried out with 100% methanol and then
with 70% methanol. Both the extracts were made solvent free by evaporation using a Butchi Type Rotary
evaporizer under reduced pressure to yield concentrated extracts. The percent yield was 2.4% for 100%
hexane extract, 4.1% for 100% chloroform extract, 5% for 100% methanol extract and 5.8% for 70%
methanol extract.
Free Radical Scavenging Activity by TLC- DPPH Method (Semi-quantitative Assay)
Thin layer chromatography was used to determine the capacity of scavenging of the free radical DPPH by
the plant extracts as described by Kwape and Chaturvedi (2011). First of all, grids with 10mm spacing
were made on a thin layer chromatography plate. Using a micro liter syringe, different concentrations of
the extracts/ standard (Gallic acid) ranging from 0.5-20mg/ml were spotted on different labeled grids. For
the control, methanol was used. The volume of the spoted samples was kept constant for the different
extracts. The spots were allowed to dry for at least 2 hours before spraying with 0.02% DPPH solution. A
photograph of the chromatogram was taken after 30 minutes. The inhibition of DPPH by different extract
was noted.
Spectrophotometric Measurement of Free Radical,2, 2- diphenyl-1-picryl Hydrazyl Radical (DPPH)
Scavenging Activities of Extracts by using
For the determination of scavenging properties of different extracts of SR using DPPH (2, 2-diphenyl-1picrylhydrazlyl), the procedure was carried out as described by Yeboah and Majinda (2009). Different
concentrations of Gallic acid or plant extracts were prepared ranging from 0.5- 5mg/ml. 0.02% of DPPH
was prepared and stored in the refrigerator until ready for use. One milliliter of the plant extract or
standard was added to an equal volume of DPPH solution. A control mixture containing 1ml of the
solvent and DPPH was prepared and all the mixtures were kept in darkness for 30 minutes, hence
absorbance was measured at 517nm using methanol as the blank. Using the equation 1 below, inhibition
curve versus concentration was plotted, and the concentration of each sample required to reduce DPPH
radical by 50% was extrapolated.
I= {(Absorbance control – Absorbance sample)/ Absorbance control} * 100
2, 2-Azobis-3-ethyl Benzothiazoline-6-sulphonic Acid (ABTS) Radical Scavenging Activity
The antioxidant capacity was estimated in terms of the ABTS+ radical scavenging activity following the
procedure described by Pellegrini et al., (1999). To obtain ABTS+ radical, 7 mM ABTS stock solution
was incubated with 2.45 mM potassium persulfate in the dark at room temperature for 16 hours before
use. The ABTS+ radical solution was diluted with distilled water to an absorbance of 1.000. Inhibiting
concentrations of extracts were tested at 6.25, 12.5, 25, 50 and 100µg/ml. Gallic acid was tested at 1.56,
3.13, 6.25, 12.5 and 25µg/ml.
© Copyright 2014 | Centre for Info Bio Technology (CIBTech)
6
Indian Journal of Plant Sciences ISSN: 2319–3824(Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm
2015 Vol.4 (1) January-March, pp.5-10/Chaturvedi et al.
Research Article
The ABTS+ radical-scavenging activity of the samples was expressed as the percent inhibition of the free
radical by using following equation:
% inhibition =[(Absorbance of control – Absorbance of test sample)/Absorbance of control*100
The concentration of each sample required to reduce ABTS radical by 50% (I50) was also determined.
Determination of the Phenol Content
Method described by Stoilova et al., (2007) was used to measure the total phenolic content of all four
extract using Folin-Ciocalteu reagent. Gallic acid was used as standard. Folin Ciocalteu reagent was
diluted 10 times with distilled water. 0.1 ml Gallic acid or SR (0-50mg/ml) were mixed with 0.75 mL of
the diluted Folin–Ciocalteu reagent and incubated at room temperature. Then, 0.75 mL of 2% sodium
carbonate solution was added to the mixture. The mixture was allowed to stand in darkness for an hour
before measuring the absorbance at 765 nm using a UV–Visible spectrophotometer. Total Phenol Content
values were determined from a calibration curve prepared with a series of Gallic acid standards. Results
are expressed as mg of Gallic acid equivalents/g fresh weight (mg GAE/g).
RESULTS AND DISCUSSION
Results
TLC/DPPH Antioxidant Activity of Different Extracts of SR
Results of TLC/DPPH semi quantitative assay are shown in photograph of TLC plate after
experimentation (Figure 1). Various concentrations (0.5mg/ml to 20mg/ml) of whole plantextracts from
different solvents spotted on the TLC sheet showed high activity, indicated by theyellow color over the
purple DPPH background. The extentof yellow color showed the antioxidant activities of the extracts
against DPPH free radicals. The TLC sheet showed the highest activities with 70% methanol extract
followed by 100% methanol and 100% chloroform extract. The extent of yellow coloration produced by
scavenging DPPH free radicals was comparable with yellow color produced by the activities of Gallic
acid. 100% hexane extract produced the least effect.
Figure 1: Free radical scavenging activity by semi quantitative method (after 30 minutes of
spraying with DPPH)
© Copyright 2014 | Centre for Info Bio Technology (CIBTech)
7
Indian Journal of Plant Sciences ISSN: 2319–3824(Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm
2015 Vol.4 (1) January-March, pp.5-10/Chaturvedi et al.
Research Article
Spectrophotometric Assay of DPPH Radical Scavenging Activities of Different Extracts
Results of Quantitative measurement of DPPH radical inhibition by different types of SR extracts
presented in Figure 2. It indicated that the inhibition activity was concentration dependent. The extent of
percentage inhibition also increased from a low polarity solvent extract to a high polarity solvent extract.
Thus the inhibition showed the pattern of 70% methanol > 1000% methanol> 100% >100% hexane
respectively. The standards Gallic acid displayed 100 % DPPH inhibition at the dose 5000ug/ml.
120
100
% inhibition
80
Hexane
60
Chloroform
Methanol
40
70% Methanol
Gallic acid
20
0
500
1000
3000
5000
Cocentration µg/ml
Figure 2: Percent DPPH inhibition versus concentration of different SR extracts
Phenol Content
As presented in Table 1, the highest amount of phenol content was present in 70% methanol extract (562
mg GAE/g)) followed by 100% chloroform and 100% methanol extract. Least amount was present in
100% hexane extract (230mg GAE/g).
Table 1: Total phenol content of SR
Plant extract
Total phenol content(mg GAE/g)
70% methanol
562±2.4
100% methanol
520±3.1
100% chloroform
466.6±6.7
100% hexane
230±2.8
ABTS Radical Scavenging Activity
Table 2 showed the results of ABTS free radical scavenging activities of different SR extracts. Effect of
ABTS free radical scavenging activity of 70% methanol, 100% methanol, 100% chloroform and 100
%hexane extracts of SR was tested at varying concentrations of 6.25, 12.5, 25, 50 and 100µg/ml. The
method was based on the ability of antioxidant molecules to quench the long lived ABTS radical action.
Significant ABTS IC50 was determined as shown in the table.Highest ABTS scavenging activity was
seen inmethanol extract having IC50 (µg/ml)of 50. Gallic acid showed IC50 (µg/ml) value of 25.
© Copyright 2014 | Centre for Info Bio Technology (CIBTech)
8
Indian Journal of Plant Sciences ISSN: 2319–3824(Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm
2015 Vol.4 (1) January-March, pp.5-10/Chaturvedi et al.
Research Article
Table 2: In vitro ABTS free radical scavenging activity of different extracts of SR.
Plant part
Tested extract
% inhibition
IC50 (µg/ml)
Whole plant extract
Reference standard
70% methanol
100% methanol
Chloroform
Hexane
Gallic acid
63.8
66.7
55.1
51.5
94.4
50
50
100
100
25
Discussion
Above results indicate that all the extracts of aerial parts of SR have antioxidant properties. Any
substance can be considered as anti-oxidant if it scavenges free radicals. DPPH is considered as proton
free radical and scavenging of proton free radical is one of the mechanisms to reduce oxidants in the
biological system (Krystyna and Anna, 2013). In the present study, different extracts of SR scavenged
DPPH radicals at different degrees. As per TLC-DPPH study, 70% methanol extract has shown maximum
scavenging followed by 100% methanol extract. At the dosage, 500 and 1000ug/ml, 100% methanol
extract shows higher inhibition of DPPH radical than 100% chloroform extract. But at the dosage
3000ug/ml, both the extracts show similar inhibition. Hexane extract has shown least scavenging. This
result is further supported by quantitative measurement of percent inhibition of DPPH radical where
highest inhibition was shown by 70% methanol extract and least inhibition by 100% hexane extract. Thus
Percentage inhibition increased from a low polarity solvent extract to a high polarity solvent extract. In
ABTS assay method, ABTS reacts with potassium persulphate to produce ABTS + which gives a blue
green chromogen.
Antioxidant reductant, the blue color becomes colorless the absorbance of which is measured at 734 nm.
In the present study, the percentage inhibition of ABTS + radical by different extracts showed the same
pattern like DPPH scavenging. Here 70 methanol extract shows maximum inhibition followed by 100%
methanol extract, 100%chloroform extract. The anti-oxidant effects of plants are attributed in part,
because of their phenol content. These compounds interrupt the propagation of chain reaction of lipid peroxidation by contributing a hydrogen atom with the formation of relatively stable compound that prevents
the propagation of oxidation process. Here in this study, 70% methanol extract has the highest phenol
content and hexane extract had least amount of phenol content. High content of phenol content could be
the reason for high free radical scavenging activities of all extracts.
ACKNOWLEDGEMENT
Authors are thankful to the Office of Research and Development (ORD), University of Botswana for
providing funds to carry this work.
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Indian Journal of Plant Sciences ISSN: 2319–3824(Online)
An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm
2015 Vol.4 (1) January-March, pp.5-10/Chaturvedi et al.
Research Article
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